Highly-compressible fluid chromatography is a type of chromatography that is configured to operate with a solvent that includes a fluid (e.g., carbon dioxide, Freon, etc.) that is in a gaseous state at ambient/room temperature and pressure. Typically, highly-compressible fluid chromatography involves a fluid that experiences noticeable density changes over small changes in pressure and temperature. Although highly-compressible fluid chromatography can be carried out with several different compounds, in the current document CO2 will be used as the reference compound as it is the most commonly employed. (It is noted that highly-compressible fluid chromatography has also been referred to as CO2-based chromatography, or in some instances as supercritical fluid chromatography (SFC), especially where CO2 is used as the mobile phase. In this application, mobile phase is used as a term to describe the primary source of a combined flow stream flowing through a chromatography column. For example, in a separation in which CO2 and methanol (a co-solvent) are mixed together to create a combined flow stream passing through a chromatography column, the term mobile phase will refer to the CO2 and the methanol will be referred to as a co-solvent. The combined mobile phase and co-solvent will be referred to as mobile phase co-solvent mixture or mobile phase solution.)
Highly-compressible fluid chromatography combines many of the features of liquid chromatography (LC) and gas chromatography (GC), and can often be used for separations with compounds that are not suitable either for LC or GC. For example, CO2-based chromatography can be advantageous for separation and analysis of hydrophilic and chiral compounds, lipids, thermally-labile compounds and polymers. Other advantages include faster separations, and lower cost and toxicity of the mobile phase, when using CO2 as a solvent, compared to many liquid mobile phases typically used in LC. In a highly-compressible fluid system, typically a smaller quantity of organic solvent, or even no organic solvent, may be used, and as a result a concentrated solution of a compound can be obtained without the need to evaporate a large quantity of solvent in the eluent.
Additionally, certain analytical samples may exhibit higher solubility in highly-compressible fluids, or in a mixture of a highly-compressible fluid and co-solvents, than in the liquid mobile phases typically used in LC. This increased solubility is applied to extractions in a range of settings, from the research laboratory to industrial coffee decaffeination, generally under the name of super-critical fluid extraction. In either setting, the highly-compressible fluid can improve the ability of the system to dissolve the sample.
In addition to carbon dioxide, a liquid organic co-solvent is typically added. The co-solvent may also be referred to as a modifier; the terms are used interchangeably herein. A common co-solvent is methanol. Examples of other co-solvents include acetonitrile and alcohols such as ethanol and isopropanol. The CO2 mobile phase and co-solvent (if any) mixture is maintained at a pressure and temperature where the mixture remains as a homogeneous, single phase. To do so, systems must be able to provide and maintain tight control over temperature, pressure, etc. In systems where the sample is dissolved in an organic solvent for injection, frequently, but not necessarily, the same solvent will be used as the co-solvent.
One factor that influences the separation power of any chromatographic system is the efficiency of the system, which is reduced by band broadening or band dispersion produced by the system. The terms “band broadening” and “band dispersion” are used interchangeably herein. Brand broadening negatively affects efficiency, as a result, a reduction in band broadening will improve the separation power of an instrument.
Extra-column band broadening (i.e., band broadening attributed to system components lying outside of the column) can occur in a chromatography system due to various factors. For example, upstream of the column, dispersion can occur after the band leaves the injector, while it is traveling towards the column inlet. An ideal sample leaves the injector as a rectangular band 10 in a conduit 12, e.g., as shown in FIG. 1A. After the sample band leaves the injector, the band is transported from the injector to the column inlet. The diffusivity of analytes in the mobile phase, the co-solvent, and in the mixture of the two determines dispersion while the band travels along the tubing connecting the injector to the column inlet—higher diffusivity and increased connecting tube volume and number of other connectors contribute to increased band broadening. For example, FIG. 1B illustrates a diffused sample band 14 in a conduit 12. Analyte diffusivity in typical highly-compressible fluid mobile phases, such as CO2, is generally greater than in solvents used in conventional LC, which could result in a diffused band at the column inlet. Another factor that can affect dispersion inside the column is a mismatch between the composition of the sample solvent and the mobile phase. For example, severe band distortion leading to separation loss can take place if a sample is prepared in a solvent having a composition markedly different than the composition of the mobile phase. See Mishra M, Rana C, De Wit A, Martin M., Influence of a strong sample solvent on analyte dispersion in chromatographic columns, J Chromatogr A. 2013 Jul. 5; 1297:46-55. Another factor that can lead to band broadening is additional volume added to a system outside the column, i.e., adding multiple fluidic lines, components (e.g., mixers) or connectors.
In conventional CO2-based chromatography preparative systems, there are two commonly used techniques for injecting sample/feed solution into the mobile stream. See Arvind Rajendran, Design of preparative supercritical fluid chromatography, J Chromatogr. A., 2012 Jun. 7; 1250:227-249. The first conventional technique (illustrated in FIG. 2), which is also commonly used in HPLC, injects the feed solution directly into the CO2 plus co-solvent/modifier mixture. For this technique, the feed solution is generally prepared by dissolving the sample in the co-solvent to permit injection. This technique, however, can lead to significant distortion of the chromatographic band even when injecting moderate volume of the feed solution. This is because the solvent used to prepare the feed solution will only be the organic solvent, leading to significant mismatch in feed solvent versus mobile phase and co-solvent mixture. The second technique (illustrated in FIG. 3), which is used to address mismatch, is to inject the sample directly into the co-solvent before the co-solvent is mixed with the CO2. Here too, the sample is introduced to the chromatography system by addition to co-solvent only, and the amount of sample that may be added is thus limited by the dissolving power of the co-solvent. This technique has further limitations due to problems associated with mixing of the sample/feed solution with co-solvent. That is, the mixing process can significantly distort the feed band profile, resulting in extra-column band dispersion, which can lead to overlapping peaks inside the column resulting in yield loss, especially for close-eluting analytes, or impurities.
Additionally, when the feed solution is prepared without CO2, the solubility of the sample in the feed solution may be different from the solubility in the mobile phase and co-solvent mixture. This difference may cause problems within the system: if the solubility is greater in the mixture, then operating the system at the solubility associated with the feed solution will result in a lowered concentration than could be obtained; conversely, if the sample solubility is lower in the feed solution, when the feed solution is introduced into the mixture, the sample may crystallize, or crash-out of solution.
Another approach to address solubility problems is to provide an extraction injection device. Such a system, as shown in FIGS. 4A, 4B, and 4C, uses an extraction vessel connected to the co-solvent and mobile phase pumps to allow for solubilizing the feed/sample material inside the extraction vessel. Once solubilized, the extraction vessel is used for injection into the chromatography system. While this method reduces the amount of precipitation, operators have less control over feed injection (e.g., varying injection amounts) as there is limited control over the flow from the extraction vessel. Additionally, the flow from the extraction vessel may exhibit inconsistent sample concentration across the flow.
Accordingly, there remains a need for a sample extraction device that integrates the extraction process into the chromatography system with controlled sample injection.